(1) Summary of the Invention
The present invention relates to a unique atypical culture of Bacillus subtilis which is used to preserve materials containing, or exposed, to undesired microorganisms. In particular, the present invention relates to a culture isolated from Pozol which inhibits a wide variety of gram-positive and gram-negative bacterium, yeasts and molds.
(2) Description of Related Art
Foods and feeds are particularly labile to microbial deterioration, and can serve as vehicles for transmission of pathogens, food-borne infections and intoxications. The microorganisms involved include bacteria, yeasts, molds and viruses. Some examples of food and feed-borne pathogens are for instance Staphylococcus aureus, Salmonella spp., Clostridium spp., Listeria monocytogenes, Yersinia spp., Vibrio spp., Escherichia coli serotype O157:H7, and Shigella spp. Enterotoxins produced by staphylococci, clostridia, and mycotoxins secreted by molds are also transmitted through foods and feeds.
Several different approaches are used to prevent microbial deterioration of foods and feeds, and transmission of microbial pathogens and/or their toxins through foods and feeds. Most of these strategies include using adequate processing methods and avoiding and preventing post-process contamination. Supplementing these steps are the use of preservatives, packaging techniques, such as vacuum shrinking films, and modified atmosphere packaging. Recently there has been an increased interest in searching for "natural inhibitors" of unwanted flora in foods and feeds. These inhibitors have included using antagonistic "safe" microorganisms such as lactic acid bacteria, microbial metabolites, such as organic acids, bacteriocins, and certain naturally derived components like spice and plant extracts.
The health promoting and antibacterial properties of a fermented Mexican drink, Pozol, have been recognized by the Mayan civilization for centuries. Pozol is made by dissolving fermented nixtamalized corn (maize) flour dough or masa in water. Only recently, have there been careful microbiological studies on this fermented native drink. The microflora of Pozol is quite complex. Wacher et al. (World J. Microbiol. Biochem. 9:269-274 (1993)) and Nuraida et al. (World J. Microbiol. Biochem. 11:567:571 (1995)) reported that the predominant microflora of Pozol consisted of lactic acid bacteria (lactobacilli, lactococci, and leuconostocs), aerobic catalase-positive mesophiles, members of the Enterobacteriaceae family, yeasts (predominantly Geotrichum candidum) and molds.
Earlier studies by Mexican researchers showed that Pozol contained Agrobacterium azotophilum (Ulloa and Herrera. Rev. Latin-amer. Microbiol. 14:15-24 (1972)), which showed a wide antibacterial activity (Herrera and Ulloa. Rev. Latin-amer. Microbiol. 17:143-147 (1975)). The Agrobacterium azotophilum isolate was a Gram-negative, non-spore forming, coccoid-bacillus, which was capsulated, and motile (Ulloa and Herrera. Rev. Latin-amer. Microbiol. 14:15-24 (1972)). They also noted that the bacterium inhibited the growth of several Gram-positive and Gram-negative bacteria, yeasts and molds. Later studies by others also attributed the antimicrobial properties of Pozol to isolates similar to Agrobacterium azotophilum (Sanchez-Fernandez and McKay. Abst. IFT Annu. Meet. Food Expo (1994)). This was a misidentification of the bacterium involved and in fact it is the strain of the present invention.
There are previous reports in the literature describing the inhibitory properties of Bacillus subtilis strains isolated from other habitats. Wilson et al., (SIM News 46:237-242 (1996)) referred to a Bacillus subtilis isolate B-3, that was effective in controlling brown rot of peaches, and was tested successfully on a semi-commercial basis in packing houses (Pusey et al., Plant Disease 72:622-626 (1988)). This strain was patented in 1984 (Pusey, L., and Wilson, C. L., U.S. Pat. No. 4,764,371). The principal agent involved in the antagonistic activity of the strain B-3 against brown rot pathogen of peaches, was found to be an antibiotic, iturin, secreted by the Bacillus (Gueldner et al., J. Agric. Food Chem. 36:366-370 (1988)). Asaka and Shoda (Appl. Environ. Microbiol. 62:4081-4085 (1996)) described the use of a strain of Bacillus subtilis, designated RB14, for controlling damping-off in tomato seedlings caused by a mold. This strain was shown to be active against several phytopathogens in in vitro studies. This strain produced two antibiotics, iturin A and surfactin, which were found in cell-free culture supernatants. Asaka and Shoda also cited other investigations, where Bacillus subtilis strains have been used to control phytopathogens in plant growth studies. No application studies on the use of microorganisms from Pozol to control unwanted flora in foods or feeds or other systems have been reported. Other U.S. patents of interest are U.S. Pat. Nos. 5,218,101 to Hansen and 5,516,682 to Hansen which relate to mutant subtilins.